The present invention relates specifically to the isolation of a yeast gene regulatory sequence (promoter), which is native to Schwanniomyces castellii and can regulate gene expression in a heterologous yeast host using starch as the sole carbon source. More specifically, the starch can be used as an inducing agent for the expression of native or foreign genes, which are fused to the isolated yeast promoter. The transformed host cells bearing the promoter-gene fusion can grow in culture medium containing various carbon sources, and the gene expression is induced by starch addition as a gene expression inducing-agent. The heterologous host is preferably bacteria, yeast, mold, plant cell, plant tissue, and whole plant.
Natural yeast strains have been identified that can use starch as a primary growth substrate via complete or partial enzymatic hydrolysis. These yeast strains include but are not limited to Saccharomycopsis fibuligera, Schwanniomyces castellii, and Saccharomyces diastaticus, which can produce and secrete both alpha-amylase and glucoamylase to, liquefy and hydrolyze starch into glucose. A fusion yeast cell strain of Saccharomyces diastaticus and Saccharomyces cerevisiae could degrade 60% of starch present in culture media within two days. In addition, other natural Saccharomyces species can ferment starch and dextrin to ethanol, as well as improve ethanol production from starch and higher sugars.
The ability to genetically modify yeast strains has greatly advanced both protein expression engineering and metabolic engineering for the past two decades. The use of yeast for producing transgenic prokaryotic and eukaryotic heterologous proteins has the added advantage that yeast and filamentous mold are microbial eukaryotes, and they are more closely related to animal cells. Hence, yeasts possess the molecular genetic manipulation and growth characteristics of prokaryotic organisms, together with the subcellular machinery for performing eukaryotic post-translational protein modification. For example, Pichia pastoris is able to synthesize functional recombinant protein and its glycosylation abilities are very similar to those of animal cells, though the glycosylation in another yeast strain, Saccharomyces cerevisiae, is different from that of an animal. In addition, the metabolic pathway of a regular ethanol producing yeast strain can be genetically altered to accumulate large amounts of lactic acid and to increase xylose utilization rate. However, only a few yeast systems (transformation vector and promoters) are available for protein engineering and metabolic engineering, which include Saccharomyces cerevisiae, Pichia pastoris, among others.
Starch utilizing yeast strain, Schwanniomyces castellii or Schwanniomyces occidentalis, is one of the most important microorganisms, since it can directly use starch as its growth medium. Due to the low level of glycosylation and the ability of protein secretion, Schwanniomyces castellii is a promising host for heterologous protein expression. However, the molecular study of Schwanniomyces sp. is very limited. Only about 21 genes have been cloned, and very few promoter sequences have been cloned and characterized in their full length from Schwanniomyces sp. The ability to genetically manipulate Schwanniomyces sp. depends on the successfulness in developing the transformation methods and gene expression systems. To effectively direct the transcription or expression of an interested gene, strong gene regulating elements or promoters are required. These promoters can be isolated from the upstream sequences of strongly expressed gene clones.
Alpha-amylase, a 56-kDa protein, is one of the highly expressed clones in Schwanniomyces castellii, and different carbohydrates such as starch regulate its expression. The expression level of alpha-amylase can be increased about 100 times when the glucose is depleted in the culture medium. The gene regulatory element (promoter) of the alpha-amylase gene would be a useful genetic element to be used for the regulation of foreign gene expression. However, the full alpha-amylase promoter of Schwanniomyces castellii has never been sequenced and characterized. To genetically manipulate Schwanniomyces sp, either for the purpose of metabolic pathway modification, conferring necessary traits such as chemical production, or producing biocatalyst of interest, high levels of mRNA expression are always desirable. Therefore, there is a need to isolate strong promoter sequences of Schwanniomyces sp. and characterize its function.
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The present invention provides the promoter clone discovery and isolation of alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate higher gene expression in starch culture medium.
An object of the present invention is to provide an isolated yeast promoter, which is native to Schwanniomyces castellii (ATCC 26077) and located upstream of and in control of an alpha-amylase gene.
Another object of the invention is to provide an isolated yeast promoter that has a sequence of 1554 base pairs prior to the initiation codon of alpha-amylase gene.
Yet another object of the invention is to provide a strong gene promoter that allows effective direction of transcription or expression of a gene of interest.
Another object of the invention is to provide a process of expressing a gene of interest in bacterial, yeast, mold, and plant/plant cell species.